A variety of different hosts including plants, algae, fungi, stramenopiles and yeast are being investigated as means for commercial polyunsaturated fatty acid [“PUFA”] production. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to linoleic acid [LA; 18:2 ω-6] and α-linolenic acid [ALA; 18:3 ω-3] fatty acid production) can be substantially altered to result in high-level production of various long-chain ω-3/ω-6 PUFAs. Whether this is the result of natural abilities or recombinant technology, production of arachidonic acid [ARA; 20:4 ω-6], eicosapentaenoic acid [EPA; 20:5 ω-3] and docosahexaenoic acid [DHA; 22:6 ω-3] may all require expression of a Δ5 desaturase.
Most Δ5 desaturase enzymes identified thus far have the primary ability to convert dihomo-γ-linolenic acid [DGLA; 20:3 ω-6] to ARA, with secondary activity in converting eicosatetraenoic acid [ETA; 20:4 ω-3] to EPA. Numerous Δ5 desaturases have been disclosed in both the open literature and the patent literature. General characteristics of Δ5 desaturases, based on desaturase evolution, are well-described by P. Sperling et al. (Prostaglandins Leukot. Essent. Fatty Acids, 68:73-95 (2003). Along with Δ6, Δ8 and Δ4 desaturases, Δ5 desaturases are known as long-chain PUFA “front-end” desaturases (wherein desaturation occurs between a pre-existing double bond and the carboxyl terminus of the fatty acid's acyl group, as opposed to methyl-directed desaturation). These desaturases are characterized by three histidine boxes [H(X)3-4H (SEQ ID NOs:1 and 2), H(X)2-3HH (SEQ ID NOs:3 and 4) and H/Q(X)2-3HH (SEQ ID NOs:5 and 6)] and are members of the cytochrome b5 fusion superfamily, since they possess a fused cytochrome b5 domain at their N-terminus which serves as an electron donor. The cytochrome b5 domain also contains a conserved heme-binding motif (i.e., a histidine-proline-glycine-glycine sequence or “HPGG” [SEQ ID NO:180] sequence), despite divergence of the remaining cytochrome b5 domain sequences. These motif sequences are the subject of U.S. Pat. No. 5,972,664.
A number of studies have suggested that the HPGG motif is implicated in enzyme activity. Sayanova, O. et al. (Plant Physiol., 121:641 (1999)) performed site-directed mutagenesis to replace the histidine residue of the HPGG motif with an alanine residue in the Δ6 desaturase of borage. The mutant enzyme was expressed in Arabidopsis; however, no enzymatic activity could be measured, suggesting that the cytochrome b5 domain of the desaturase was important for function. A similar study was performed in a rat Δ6 desaturase, where an alanine for histidine substitution was engineered within the HPGG motif. The mutated protein also had no activity (Guillou, H., et al., J. Lipid Res., 45:32-40 (2004)). Most recently, Hongsthong, A. et al. (Appl. Microbiol. Biotechnol., 72:1192-1201 (2006)) reported substitution of the histidine residue of the HPGG motif with an alanine residue in the Δ6 desaturase of Spirulina. As with previous reports, the mutation rendered the mutant enzyme unable to produce GLA in E. coli, suggesting that the cytochrome b5 domain was important for activity and that alterations in this motif will result in diminished enzyme activity. Although Δ5 desaturase enzymes are relatively common and well characterized, there remains a need for enzymes that are efficiently expressed at high levels in production host cells capable of making PUFAs.
The problem to be solved therefore is to discover new Δ5 desaturase enzymes having high activity that are well suited for integration into PUFA biosynthetic pathways in commercially useful host cells. Applicants have solved the stated problem through the unexpected discovery that alterations in the HPGG motif of the cytochrome b5 domain of various Δ5 desaturases resulted in up to 38% improvement in enzymatic activity, based on the conversion of DGLA to ARA.